The present invention relates generally to methods for regulating processes mediated by junctional adhesion molecule (JAM), and more particularly to the use of modulating agents comprising a JAM cell adhesion recognition sequence and/or an antibody that specifically recognizes such a sequence for inhibiting functions such as cell adhesion and the formation of tissue permeability barriers.
Cell adhesion is a complex process that is important for maintaining tissue integrity and generating physical and permeability barriers within the body. All tissues are divided into discrete compartments, each of which is composed of a specific cell type that adheres to similar cell types. Such adhesion triggers the formation of intercellular junctions (i.e., readily definable contact sites on the surfaces of adjacent cells that are adhering to one another), also known as tight junctions, gap junctions, spot desmosomes and belt desmosomes. The formation of such junctions gives rise to physical and permeability barriers that restrict the free passage of cells and other biological substances from one tissue compartment to another. For example, the blood vessels of all tissues are composed of endothelial cells. In order for components in the blood to enter a given tissue compartment, they must first pass from the lumen of a blood vessel through the barrier formed by the endothelial cells of that vessel. Similarly, in order for substances to enter the body via the gut, the substances must first pass through a barrier formed by the epithelial cells of that tissue. To enter the blood via the skin, both epithelial and endothelial cell layers must be crossed.
Cell adhesion is mediated by specific cell surface adhesion molecules (CAMs). There are many different families of CAMs, including the immunoglobulin, integrin, selectin and cadherin superfamilies, and each cell type expresses a unique combination of these molecules. Cadherins are a rapidly expanding family of calcium-dependent CAMs (Munro et al., In: Cell Adhesion and Invasion in Cancer Metastasis, P. Brodt, ed., pp. 17-34, RG Landes Co., Austin Tex., 1996). The cadherins (abbreviated CADs) are membrane glycoproteins that generally promote cell adhesion through homophilic interactions (a CAD on the surface of one cell binds to an identical CAD on the surface of another cell). Cadherins have been shown to regulate epithelial, endothelial, neural and cancer cell adhesion, with different CADs expressed on different cell types. For example, N (neural)xe2x80x94cadherin is predominantly expressed by neural cells, endothelial cells and a variety of cancer cell types. E (epithelial)xe2x80x94cadherin is predominantly expressed by epithelial cells. VE (vascular endothelial)xe2x80x94cadherin is predominantly expressed by endothelial cells. Other CADs are P (placental)xe2x80x94cadherin, which is found in human skin, and R (retinal)xe2x80x94cadherin. A detailed discussion of the cadherins is provided in Munro SB et al., 1996, In: Cell Adhesion and Invasion in Cancer Metastasis, P. Brodt, ed., pp. 17-34 (RG Landes Company, Austin Tex.) and Lampugnani and Dejana, Curr. Opin. Cell Biol. 9:674-682, 1997.
CAD-mediated cell adhesion triggers a cascade of events that lead to the formation of intercellular junctions, and ultimately to the establishment of permeability barriers between tissue compartments. The intercellular junction that is directly responsible for the creation of permeability barriers that prevent the diffusion of solutes through paracellular spaces is known as the tight junction, or zonula occludens (Anderson and van Itallie, Am. J. Physiol. 269:G467-G475, 1995; Lampugnani and Dejana, Curr. Opin. Cell Biol. 9:674-682, 1997).
The transmembrane component of tight junctions that has been the most studied is occludin (Furuse et al., J. Cell Biol. 123:1777-1788, 1993; Furuse et al., J. Cell Sci. 109:429-435, 1996). This protein appears to be expressed by all endothelial cell types, as well as by most epithelial cell types. Occludin is believed to be directly involved in cell adhesion and the formation of tight junctions (Furuse et al., J. Cell Sci. 109:429-435, 1996; Chen et al., J. Cell Biol 138:891-899, 1997). A detailed discussion of occludin structure and function is provided by Lampugnani and Dejana, Curr. Opin. Cell Biol. 9:674-682, 1997.
More recently, junctional adhesion molecule (JAM) has been identified as an immunoglobulin gene superfamily member that is a component of tight junctions (Martin-Padura et al., J. Cell. Biol. 142:117-127, 1998). This protein is selectively concentrated at intercellular junctions of endothelial and epithelial cells of different origins, and has been shown to play a role in regulating monocyte transmigration.
Although cell adhesion is required for certain normal physiological functions, there are situations in which the level of cell adhesion is undesirable. For example, many pathologies (such as autoimmune diseases and inflammatory diseases) involve abnormal cellular adhesion. Cell adhesion may also play a role in graft rejection. In such circumstances, modulation of cell adhesion may be desirable.
In addition, permeability barriers arising from cell adhesion create difficulties for the delivery of drugs to specific tissues and tumors within the body. For example, skin patches are a convenient tool for administering drugs through the skin. However, the use of skin patches has been limited to small, hydrophobic molecules because of the epithelial and endothelial cell barriers. Similarly, endothelial cells render the blood capillaries largely impermeable to drugs, and the blood/brain barrier has hampered the targeting of drugs to the central nervous system. In addition, many solid tumors develop internal barriers that limit the delivery of anti-tumor drugs and antibodies to inner cells.
Attempts to facilitate the passage of drugs across such barriers generally rely on specific receptors or carrier proteins that transport molecules across barriers in vivo. However, such methods are often inefficient, due to low endogenous transport rates or to the poor functioning of a carrier protein with drugs. While improved efficiency has been achieved using a variety of chemical agents that disrupt cell adhesion, such agents are typically associated with undesirable side-effects, may require invasive procedures for administration and may result in irreversible effects.
Accordingly, there is a need in the art for compounds that modulate cell adhesion and improve drug delivery across permeability barriers without such disadvantages. The present invention fulfills this need and further provides other related advantages.
The present invention provides compounds and methods for modulating JAM-mediated cell adhesion and the formation of permeability barriers. Within certain aspects, the present invention provides cell adhesion modulating agents that inhibit or enhance JAM-mediated cell adhesion. Certain modulating agents are 4-16 amino acid peptides (which may be linear or cyclic) that comprise the sequence Asp-Pro-Lys (DPK). Within other embodiments, a modulating agent may (a) comprise at least five or seven consecutive amino acid residues of a JAM CAR sequence having the formula:
Ser-Phe-Thr-Ile-Asp-Pro-Lys-Ser-Gly (SEQ ID NO:1)
and (b) contain no more than 50 consecutive amino acid residues present within a JAM.
Within certain embodiments, a modulating agent as described above comprises a JAM CAR sequence that is present within a cyclic peptide. The cyclic peptide may have the formula: 
wherein X1, and X2 are optional, and if present, are independently selected from the group consisting of amino acid residues and combinations thereof in which the residues are linked by peptide bonds, and wherein X1 and X2 independently range in size from 0 to 10 residues, such that the sum of residues contained within X1 and X2 ranges from 1 to 12; wherein Y1 and Y2 are independently selected from the group consisting of amino acid residues, and wherein a covalent bond is formed between residues Y1 and Y2; and wherein Z1 and Z2 are optional, and if present, are independently selected from the group consisting of amino acid residues and combinations thereof in which the residues are linked by peptide bonds. In certain embodiments, Y1 comprises an N-acetyl group and/or Y2 comprises a C-terminal amide group. Y1 and Y2 may be covalently linked via any suitable bond, including a disulfide bond, an amide bond or a thioether bond.
The present invention further provides, within other aspects, polynucleotides encoding a modulating agent as provided above, expression vectors comprising such a polynucleotide, and host cells transformed or transfected with such an expression vector. Within further aspects, the present invention provides modulating agents that comprise an antibody or antigen-binding fragment thereof that specifically binds to a JAM CAR sequence as described above and modulates a JAM-mediated function.
The present invention further provides modulating agents comprising a mimetic of a JAM CAR sequence provided above, wherein the mimetic is capable of modulating a JAM-mediated function.
Within other aspects, modulating agents as described above may be linked to one or more of a drug, a detectable marker, a targeting agent and/or a support material. Alternatively, or in addition, modulating agents as described above may further comprise one or more of: (a) a cell adhesion recognition sequence that is bound by an adhesion molecule other than a JAM; and/or (b) an antibody or antigen-binding fragment thereof that specifically binds to a cell adhesion recognition sequence bound by an adhesion molecule other than a JAM. Such adhesion molecules may be selected from the group consisting of integrins, cadherins, occludin, N-CAM, claudins, PE-CAM, desmogleins, desmocollins, fibronectin, laminin and other extracellular matrix proteins.
The present invention further provides pharmaceutical compositions comprising a cell adhesion modulating agent as described above, in combination with a pharmaceutically acceptable carrier. Such compositions may further comprise a drug. In addition, or alternatively, such compositions may further comprise one or more of: (a) a peptide comprising a cell adhesion recognition sequence that is bound by an adhesion molecule other than a JAM; and/or (b) an antibody or antigen-binding fragment thereof that specifically binds to a cell adhesion recognition sequence bound by an adhesion molecule other than a JAM.
Within further aspects, methods are provided for modulating cell adhesion, comprising contacting a JAM-expressing cell with a cell adhesion modulating agent as described above.
Within one such aspect, the present invention provides methods for increasing vasopermeability in a mammal, comprising administering to a mammal a cell adhesion modulating agent as provided above, wherein the modulating agent inhibits JAM-mediated cell adhesion.
Within another aspect, methods are provided for reducing unwanted cellular adhesion in a mammal, comprising administering to a mammal a cell adhesion modulating agent as provided above, wherein the modulating agent inhibits JAM-mediated cell adhesion.
In yet another aspect, the present invention provides methods for enhancing the delivery of a drug through the skin of a mammal, comprising contacting epithelial cells of a mammal with a cell adhesion modulating agent as provided above and a drug, wherein the modulating agent inhibits JAM-mediated cell adhesion, and wherein the step of contacting is performed under conditions and for a time sufficient to allow passage of the drug across the epithelial cells.
The present invention further provides methods for enhancing the delivery of a drug to a tumor in a mammal, comprising administering to a mammal a cell adhesion modulating agent as provided above and a drug, wherein the modulating agent inhibits JAM-mediated cell adhesion.
Within further aspects, the present invention provides methods for treating cancer in a mammal, comprising administering to a mammal a cell adhesion modulating agent as provided above, wherein the modulating agent inhibits JAM-mediated cell adhesion.
The present invention further provides methods for inhibiting angiogenesis in a mammal, comprising administering to a mammal a cell adhesion modulating agent as provided above, wherein the modulating agent inhibits JAM-mediated cell adhesion.
Within further aspects, the present invention provides methods for enhancing drug delivery to the central nervous system of a mammal, comprising administering to a mammal a cell adhesion modulating agent as provided above, wherein the modulating agent inhibits JAM-mediated cell adhesion.
The present invention further provides methods for enhancing wound healing in a mammal, comprising contacting a wound in a mammal with a cell adhesion modulating agent as provided above, wherein the modulating agent enhances JAM-mediated cell adhesion.
Within a related aspect, the present invention provides methods for enhancing adhesion of foreign tissue implanted within a mammal, comprising contacting a site of implantation of foreign tissue in a mammal with a cell adhesion modulating agent as provided above, wherein the modulating agent enhances JAM-mediated cell adhesion.
The present invention further provides methods for inducing apoptosis in a JAM-expressing cell, comprising contacting a JAM-expressing cell with a cell adhesion modulating agent as provided above, wherein the modulating agent inhibits JAM-mediated cell adhesion.
The present invention further provides methods for modulating monocyte traffic in a mammal, comprising administering to a mammal a cell adhesion modulating agent as provided above.
The present invention further provides methods for identifying an agent capable of modulating JAM-mediated cell adhesion. One such method comprises the steps of (a) culturing cells that express a JAM in the presence and absence of a candidate agent, under conditions and for a time sufficient to allow cell adhesion; and (b) visually evaluating the extent of cell adhesion among the cells.
Within another embodiment, such methods may comprise the steps of: (a) culturing normal rat kidney cells in the presence and absence of a candidate agent, under conditions and for a time sufficient to allow cell adhesion; and (b) comparing the level of cell surface JAM and E-cadherin for cells cultured in the presence of candidate agent to the level for cells cultured in the absence of candidate agent.
Within a further embodiment, such methods may comprise the steps of: (a) culturing human aortic endothelial cells in the presence and absence of a candidate agent, under conditions and for a time sufficient to allow cell adhesion; and (b) comparing the level of cell surface JAM and N-cadherin for cells cultured in the presence of candidate agent to the level for cells cultured in the absence of candidate agent.
Within yet another embodiment, such methods comprise the steps of: (a) contacting an antibody that binds to a modulating agent comprising a JAM CAR sequence with a test compound; and (b) detecting the level of antibody that binds to the test compound.
The present invention further provides methods for detecting the presence of JAM-expressing cells in a sample, comprising: (a) contacting a sample with an antibody that binds to a JAM CAR sequence under conditions and for a time sufficient to allow formation of an antibody-JAM complex; and (b) detecting the level of antibody-JAM complex, and therefrom detecting the presence of JAM-expressing cells in the sample.
Within further aspects, the present invention provides kits for detecting the presence of JAM-expressing cells in a sample, comprising: (a) an antibody that binds to a modulating agent comprising a JAM CAR sequence; and (b) a detection reagent.
The present invention further provides, within other aspects, kits for enhancing transdermal drug delivery, comprising: (a) a skin patch; and (b) a cell adhesion modulating agent, wherein the modulating agent comprises a JAM CAR sequence, and wherein the modulating agent inhibits JAM-mediated cell adhesion.
These and other aspects of the invention will become evident upon reference to the following detailed description and attached drawings. All references disclosed herein are hereby incorporated by reference in their entirety as if each were individually noted for incorporation.